A novel dextransucrase gene, DSRN, was obtained by ultrasoft X-ray treatment of the DSRB742 gene. The DSRN gene was further mutated via site-directed mutagenesis producing four mutants: DSRN1 (F196S), DSRN2 (Y346N), DSRN3 (K395T) and DSRN4 (P980T). Dextransucrases derived from DSRB742 and its mutants were expressed in E. coli and affinity-purified using dextran to give 80% purity. They had specific activities of 0.6–17 U/mg with Km values of 18–88 mM. DSRB742 had the lowest (0.02 s−1 · mM−1) and DSRN1 had the highest (0.13 s−1 · mM−1) Kcat/Km values. DSRN3 had the highest enzymatic transglycosylation efficiency with maltose (63% of theoretical), gentiobiose (39%), or salicine (40%).